Journal: bioRxiv
Article Title: Quantitative extrapolation from single-tags (QuEST) immunofluorescence microscopy to derive TCR signalosome stoichiometries in human primary T cells
doi: 10.64898/2026.03.28.715001
Figure Lengend Snippet: a , Schematic illustrating the principle of QuEST for quantifying molecule numbers and surface densities. b , Isolation and detection of fluorescence-labelled single molecules by TIRFM. c , Workflow for quantifying single-molecule fluorescence intensity. d , e , Intensity distribution within a 5×5-pixel region ( d ) and corresponding mean fluorescence intensity ( e ) of single CD45 AF405, UCHT-1 AF488, ICAM-1 AF568, and UCHT-1 AF647 molecules. f , Workflow for quantifying fluorescence intensity of multiple molecules. g , Quantification of UCHT-1 and ICAM-1 densities on supported lipid bilayers (SLBs) containing different percentages of Ni–NTA using QuEST. h , i , Schematic ( h ) and quantification results ( i ) determining the linear dynamic range of DOPE with different fluorescence levels. j , Linear density ranges of UCHT-1 and ICAM-1 under different fluorescence conditions. k , T cells clustering UCHT-1 and ICAM-1 on SLBs, with dynamic molecular densities quantified. l , Relative errors in quantifying UCHT-1 and ICAM-1 densities using QuEST. Data in g and i are presented as mean ± SD; data in e and k are presented as mean ± SEM. Statistical significance was assessed using an unpaired two-tailed Student’s t -test (**** P ≤ 0.0001; ns, not significant). In e , data were obtained from 16 single molecules. In g and i , data were obtained from three independent TIRF images. The structure of UCHT-1 was derived from PDB entry 3FZU, and the structure of ICAM-1 was generated using AlphaFold3 from the corresponding protein sequence.
Article Snippet: Unlabelled primary antibodies : anti-human CD8α antibody (Cell Signaling Technology, Cat#85336), anti-human CD28 antibody (Cell Signaling Technology, Cat#38774S), anti-human CD45 antibody (Cell Signaling Technology, Cat#13917S), anti-human PD-1 antibody (Cell Signaling Technology, Cat#86163T), anti-human Lck antibody (Cell Signaling Technology, Cat#2787S), anti-human ZAP-70 antibody (Cell Signaling Technology, Cat#3165S), anti-human LAT antibody (Cell Signaling Technology, Cat#45533S), anti-human PLCγ1 antibody (Cell Signaling Technology, Cat#5690S), and anti-human phospho-ZAP-70 (Tyr319) antibody (Cell Signaling Technology, Cat#2701).
Techniques: Isolation, Fluorescence, Two Tailed Test, Derivative Assay, Generated, Sequencing
